Legionella pneumophila, the causative agent of Legionnaires' Disease, is an intracellular pathogen which reproduces within human macrophages and within amoebae in most fresh water supplies. To better understand its life cycle and mode of infectivity, as well as its relationship to other intracellular parasites, its complete genome sequence will be determined, and all its putative genes identified. Sequencing will be accomplished by a mixed BAC shotgun/whole genome shotgun strategy. Gene functions will be predicted by advanced computational methods, including phylogenetic comparisons. The genes involved in L. pneumophila's intracellular lifestyle will be compared in various Legionella species and L. pneumophila subspecies to determine potential functional motifs in these genes. The expression pattern of these genes will also be compared during different stages and modes of growth (e.g. symbiotic versus vegetative). This will be accomplished by northern analysis or the use of bifunctional hybrids with reporter gene tags. Finally, allelic exchange will be used to produce null mutations in some genes of particular interest to determine their functions with regard to the Legionella life cycle and the pathogenesis of Legionnaires' Disease. The sequence of Legionella will be a valuable resource for researchers in the field. Analysis of the complete Legionella sequence should provide important information on the evolution and ecology of this organism, especially when compared to that of other bacteria already or in the process of being sequenced. It will be especially useful to compare its genome with those of Chlamydia, Rickettsia, and Mycobacteria, or viruses such as HIV, which are also intracellular pathogens. Finally, information obtained in the course of this work should be useful in designing therapeutic strategies for Legionnaires' Disease, which is fatal in a significant percentage of antibiotic-treated infected individuals.